Download Cajal`s debt to Golgi

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Review
Cajal's debt to Golgi
Edward G. Jones
University of California, Center for Neuroscience, 1544 Newton Court, Davis CA 95618, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Accepted 13 April 2010
Available online 18 April 2010
Keywords:
Cerebral cortex
Cerebellum
Retina
Golgi technique
Axon collateral
Dendrite
Neural circuitry
Contents
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
We are accustomed to thinking of Camillo Golgi and Santiago
Ramón y Cajal as contemporary scientists at war over the
neuron doctrine. This is certainly how Cajal in his biography
and later writings portrayed their relationship and Golgi did
not help matters by his most unfortunate Nobel acceptance
speech of 1906 (Golgi 1907) in which he emphasized in a
contentious way his continuing belief in an outmoded view of
the nervous system. Cajal's speech (Cajal, 1907), by contrast,
was of a kind typical of that of any modern neuroscientist in
which he outlined his past achievements in neurohistology
and then proceeded to describe his ongoing experiments in
nerve regeneration. The contrast between the two speeches is
a reflection of the fact that Golgi's work on the organization of
the central nervous system had essentially ended by 1883
when he turned mainly to investigations of malaria, while
Cajal's which had commenced only in 1888 was still in full
flight. As neuroscientists, therefore, they cannot be seen as
contemporaries. In what follows, I shall attempt to present the
case that Golgi's observations, made on tissue stained by his
black reaction represented a fundamental breakthrough in the
way in which the nervous system was viewed and that his
observations provided a firm basis upon which Cajal was later
able to build. While it would be wrong to say, as some have,
that Cajal stood on the shoulders of Golgi, there can be little
doubt that a number of Golgi's observations on the structure
and organization of nerve cells not only transformed the way
E-mail address: [email protected].
0165-0173/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainresrev.2010.04.005
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Fig. 1 – Drawings of Purkinje cells from the human cerebellum,
showing the extent of detail that could be visualized in
material fixed in chromium or osmium salts and stained with
carmine. From Kölliker (1863).
in which contemporary scientists perceived nerve cells but
also represented the starting points for Cajal's work in which
in a few years he unraveled the intrinsic circuitry of just about
every region of the nervous system.
Prior to Golgi's discovery of the black reaction (Golgi, 1873)
and his publication of the first images of nerve cells obtained
with it, knowledge of the form of nerve cells was remarkably
primitive. Purkinje did not visualize the cells that now bear his
name as the magnificent structures with their elaborate planar
set of dendrites that we know today but as no more than
globules that represented only the cell body. The later introduction of chromic acid as a fixative and carmine as a stain
extended somewhat the knowledge of the nerve cell, notably in
the hands of Deiters who identified branching dendrites (which
he called protoplasmic processes) and the axon (called the axis
cylinder). But the forms of the dendritic trees demonstrated by
Deiters and others were incomplete and the collateral branches
of axons had not been discovered. Kölliker's (1863) drawings of
Purkinje cells (Fig. 1), while definitely an advance on what
Purkinje himself had seen, are still primitive when compared
with what we have subsequently learned about the structure of
that neuron. Prior to his discovery of the black reaction, Golgi's
drawings of nerve cells which he had observed in tissue fixed in
potassium dichromate, chromic or osmic acid (Fig. 2) are little
different from those of other contemporary histologists, sometime showing branching dendrites but often with no more than
stumps of dendrites emerging from the soma and perhaps the
axon hillock and initial segment.
The first images of neurons impregnated with the Golgi
stain heralded the beginning of a revolution in how nerve
cells were viewed. Those early drawings of Golgi (e.g. Golgi,
1875) present neurons for the first time in the form in which
we are still accustomed to portraying them (Fig. 3). Golgi's
disappointment at the slow recognition in print of the
importance of his findings was real but it seems clear that
this was less on account of disbelief than on the fact that few
scientists could successfully employ his method to obtain
similar results. It was only after Cajal much later, in 1888 and
1889, applied the stain in repeated impregnations, with
longer immersion times and more concentrated reagents,
and in infant animals in which myelination was less
advanced than in adults, that others were able successfully
to use it (DeFelipe and Jones, 1992). But at the time of Golgi's
first publications no one with any eye for the nervous system
could fail to appreciate the manner in which his stain had
dramatically extended anatomical knowledge of the nerve
cell, its dendrites and its axon. Outside the world of scientific
publishing there were many who early on recognized that a
revolution had occurred and attempted to implement the
Golgi methods themselves. Fritjof Nansen in 1887, traveled to
Pavia to learn the secrets of the method (Jones, 1994), and Luis
Simarro who in 1887 was to give Cajal his first glimpse of a
Golgi preparation (Cajal, 1917) applied while in Ranvier's
laboratory in Paris. Neither of them published the results of
their investigations, however, and others such as Kölliker,
who also visited Pavia in 1888, after earlier correspondence
with Golgi, were unable to get the stain to work. To the
scientific establishment, the new revelations about nerve
cells seemed to have come as the result of a freakish accident
by a little known Italian. That other Italians, notably Tartuferi
in his study of the retina (1887) could make preparations as
compelling as those of Golgi made it clear that, given the
correct application of the stain, nerve cells could be revealed
in far greater detail than before. When Cajal reported his first
observations in short communications in 1888 (Cajal, 1888a,
Fig. 2 – Drawings of nerve cells made by Golgi in the years before his discovery of the black reaction. Left: pyramidal cells and
neuroglial cells from the human cerebral cortex sectioned after fixation in osmic acid. From Golgi (1871). Right: Ganglion cell
from the retina of a horse, from a whole mount fixed in potassium dichromate and osmic acid. From Golgi (1872).
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Fig. 3 – Golgi cell from the cerebellar cortex of a neonatal cat,
showing the stereotypy of cell form along with the free ending
dendrites and axon collaterals demonstrable by the Golgi
technique. The shadow across the middle of the figure is
formed by the fold in the original plate. From Golgi (1882, 1903).
b, c), he did not claim to be seeing the full extent of nerve cells
for the first time but rather that he had been able to replicate
the findings of Golgi on the cerebellum and of Tartuferi on the
retina. His interpretations of what he saw, however, were to
be quite different from those of Golgi and Tartuferi.
The facts that Golgi gave to the scientific world and that
Cajal was able so brilliantly to build on, were three. Cajal
himself makes a point of these in his early publications of
1888–1891 (Cajal, 1888a,b,c, 1889a,b), and amplified them in
his El nuevo concepto de la histolgía de los centros neviosos of 1892.
He was to repeat the debt that he owed to Golgi in this regard
throughout all of his publications up to and beyond his great
Histologie du Système Nerveux de l'Homme et des Vertébrés (Cajal,
1909, 1911). Although pointing out even in the earliest papers
that he disagreed with Golgi's portrayal of the nervous system
as an anastomotic network, and in later works disparaging
Golgi for this, he never failed to remark on how Golgi's
observations had led him to his conclusions about neuronal
connectivity.
To Cajal, there were two fundamental observations of Golgi
that led Cajal to make his new interpretations of the organization of the nervous system: (i) every nerve cell possesses an axon
that gives rise to numerous very fine collateral branches (Fig. 4);
and (ii) the dendrites, although branching extensively, do not
form a network and end freely (Fig. 5). The collateral branches of
the axon had never previously been seen and although Golgi felt
that the free ending dendrites might contact blood vessels in
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order to gain nourishment, that was of less consequence than
the lack of a dendritic network which had been emphasized by
earlier workers such as Gerlach (1872). Both of these points were
to form the foundations of all Cajal's work on the intrinsic
circuitry of the nervous system. To them it is necessary to add a
third, implicit in Cajal's writings but never stated in this form,
namely that nerve cells can have a stereotyped structure that is
defined by size and by the nature of the dendritic ramifications
and repeated in the same region of different animals and even of
different species. Without the revelations of the Golgi technique, this had hitherto been impossible fully to appreciate.
Nerve cells could now be given standardized names; some such
as the Purkinje cell or the pyramidal cell of the cerebral cortex
were already in existence but others were novel and Cajal was to
be more inventive than Golgi in this regard. Golgi's classification
of nerve cells into large type I or motor and smaller type II or
sensory cells was based on his determination of whether in the
case of the first, the axon became continuous with a myelinated
fiber of the white matter or, in the case of the second, remained
within the vicinity of the cell. But it was Golgi's commitment to
the network theory that led him into errors here. To take as an
example the cerebellar cortical cells that Cajal was later to call
basket cells, Golgi considered that their axons branched within
an anastomotic plexus of collaterals within the granule cell
layer from which axons emerged to enter the white matter. To
him, therefore the basket cells were Type II cells. But as soon as
Cajal in his first study of the cerebellar cortex in 1888 recognized
that the axons of the basket cells did not immediately lose their
individuality in breaking up into a network of branches but
ended instead around the cell bodies of Purkinje cells, he was
able to say that the only way these cells could communicate
with the white matter was indirectly via the axons of the
Purkinje cells. This was one of the revelations that led him
towards the concept of interneuronal connections by contact
and to the flow of activity from cell to cell. From this beginning
he was able to describe the intrinsic circuitry of just about every
region of the nervous system.
The secret of Cajal's success in the early phase of his career
lay as much in his choice of regions in which to work as upon his
repeated impregnation method and his choice of the brains of
small and immature animals for staining (DeFelipe and Jones,
1992). It is not without significance that the first regions that he
chose to study were the retina and the cerebellar cortex (Cajal,
1888a, b, c; Cajal, 1891) for here the stereotypy of cell types, their
arrangement in distinct layers and the evident flow of
information from input to output in each gave him the
opportunity to view them from a circuit based perspective. He
was led to these regions by the work on the retina of Dogiel
(1888) and Tartuferi (1887) who, in using methylene blue or Golgi
staining, had identified the layer specific cell types and by that of
Golgi (1882) who had done the same for the cerebellar cortex. In
recognizing first the free endings of the basket cells on Purkinje
cells in the cerebellum (Cajal 1888c) and of the bipolar cells of the
retina on the dendrites of ganglion cells (Cajal 1888a,b), Cajal
was led immediately to his view of connections by contact and
to the cellular basis of connectivity. His viewpoint was further
developed after his discovery that the granule cells of the
cerebellum gave rise to the parallel fibers which Golgi had not
seen (Cajal 1888c) and his identification of the mossy and
climbing fibers (Cajal 1889a), for their organization allowed him
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Fig. 4 – Purkinje cell from the human cerebellum, stained by the Golgi technique and showing for the first time the planar
dendritic tree and the axon collaterals. From Golgi (1882, 1903).
to plot the input–output connections of the cerebellar cortex. It
is not surprising that in the 45 papers published by Cajal
between 1888 and 1891 when the Neuron Doctrine was
enunciated by Wilhelm Waldeyer, the word “connections”
appears either in the title or as part of a lengthy subsection of
every paper (Jones, 1994).
Fig. 5 – One of Cajal's first drawings of a Golgi preparation, from the retina of a chicken, demonstrating the links made by the
bipolar cells between the photoreceptors and ganglion cells. From Cajal (1888).
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If it was the stereotyped forms and locations of nerve cells in
the retina and cerebellar cortex that gave Cajal the base upon
which to build his connectionist view of cerebral circuitry, when
he turned in 1890 to the cerebral cortex, it was the axon
collaterals that gave him a basis for building circuits therein
(Cajal, 1891). He was impressed by two types of collaterals: those
that branched off fibers in the white matter and ascended into
the cortex while the parent axon continued on in the white
matter, and those given off by cells in the cortex itself. The first
included callosal, corticocortical and thalamocortical fibers
while the second formed elements of intracortical circuitry
(Cajal, 1891, 1892, 1894, 1899a,b). In his early papers on the
cortex, Cajal can be quite tiresome in his enthusiastic descriptions of collaterals.
Golgi, in his studies on the cerebral cortex, had identified
the pyramidal cell as the source of fibers leading from cortex
to white matter and had demonstrated the presence of
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numerous intracortical collaterals on these fibers (Fig. 6).
These collaterals were to form an important element in
Cajal's thinking about cortical circuitry. In his first studies on
the cortex Cajal emphasized the pyramidal cell as the source
of output from the cortex and, on identifying the terminations
of the fibers coming from the thalamus, proceeded to
investigate how these inputs might be linked to the output
cells by the intracortical connections formed by the collaterals of pyramidal cell axons and by the short axons of
intrinsic neurons. His first proposals on this subject appeared
in the Nuevo Concepto of 1892 and in his Croonian lecture of
1894 and were elaborated further in his Textura del Sistema
Nervioso del Hombre y de los Vertebrados (Cajal, 1899, 1904) and
its French translation, the Histologie (Fig. 7) (DeFelipe and
Jones, 1988). In summarizing his views in the latter two works,
Cajal identified a series of intracortical and corticocortical
“arcs” intercalated between “the pathways of reception and
Fig. 6 – Pyramidal cells and non-pyramidal cells stained by the Golgi technique in the human cerebral cortex, showing the axon
collaterals on the pyramidal cells. From Golgi (1882, 1903).
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Fig. 7 – Early drawings made by Cajal in order to illustrate his views of the circuitry of the cerebral cortex, with his typical arrows
indicating the direction of impulse propagation from cell to cell by means of the collaterals of pyramidal cell axons. A is from his
Nuevo concepto (Cajal 1892) and B is from his Croonian lecture (Cajal 1894). In these days before his identification of the thalamic
afferents he draws the afferent fibers (E in A, G in B) as reaching layer I of the cortex. In this case he was probably visualizing
corticocortical or callosal fibers. Collaterals of the white matter are represented by F in A and I in B.
emission”, that is between the thalamocortical fibers and the
pyramidal cells. As he saw it, “the single channel represented
by an afferent fiber is broken down into an infinity of
secondary channels that traverse almost the whole gray
matter of the hemispheres along variable radii”. The first
“short or principal arc” (Fig. 8) is made up of the afferent axon
ending directly on the dendrites of pyramidal cells mainly in
the third and fifth layers, and by this direct route sensory
impulses are rapidly transformed into reflex outputs such as
movements. But the communication is not between single
elements but between a group of afferent fibers and a more
numerous constellation of pyramidal neurons. This leads to
an “avalanche of conduction” that is further amplified and
extended to other output neurons by the collateral branches
of the projection axons that spread excitation to other cells of
the same layers (Figs 7, 8). The second, “intragriseal arc with
intercalation of cells with ascending axons” (Fig. 9), begins
with terminations of the afferent fibers upon small pyramidal
and stellate cells located in layer IV (D in Fig. 7B) whose
ascending axons contact the dendritic tufts of layer III and V
pyramidal cells. The collaterals of the pyramidal cells will
carry the activity to other groups of pyramidal cells located in
the same gyrus but at a considerable distance from the
territory of arborization of the afferent fiber. This arc of
activity would be further extended by the intercalation of the
axons of horizontal cells located in layer I (F in Fig. 7B)
carrying the activity to even more distant pyramidal cells,
including those located in other gyri. The third “intraareal
intragriseal arc” (Fig. 10) is formed by pyramidal cells whose
axons terminate in the association areas of the same
hemisphere, extending the influence of the original afferent
input to areas concerned with higher level cortical function.
An extension of this arc is the “interhemispheric arc”
whereby activity is carried across the corpus callosum by
the axons of pyramidal cells.
Cajal emphasized, however that the role of the nonpyramidal cells whose short axons remained within the
cortex was more than to form a link between input fibers and
output cells. If this were so, he says, then the arrangement of
the axons of these cells would direct the afferent impulses
into many “useless detours”. Rather, he saw the short axon
cells as serving as “condensers or accumulators of nervous
energy” which would serve to amplify impulses passing
through the various arcs described above and perhaps
convert it into some kind of lingering trace that persists
after the initial activity has passed. Not a bad idea when made
in the absence of knowledge about inhibition or long term
synaptic plasticity.
Golgi's gift to Cajal, who called him without condescension
“the savant of Pavia”, cannot be underestimated and should be
viewed from a perspective that is unencumbered by their
contentious squabbling over the neuron doctrine, a controversy
that Cajal was only too eager to promote. When viewed from a
more objective perspective, Golgi must be seen as having
anteceded Cajal and to have given to the scientific world a
vision of neurons and their organization in the central nervous
B RA I N R ES E A RC H R EV IE W S 6 6 (2 0 1 0) 8 3– 9 1
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Fig. 8 – Schematic view of the first of Cajal's intracortical and corticocortical “arcs” intercalated between “the pathways of reception
and emission”. This first “short or principal arc” is made up of a thalamic axon ending on the dendrites of pyramidal cells, forming
the most direct route for transmission of sensory messages into reflex outputs such as movements. The arc is not made up of single
elements but by groups of afferent fibers connecting to groups of pyramidal neurons. This leads to an “avalanche of conduction”
that is amplified and extended to other output neurons by the collateral branches of the axons of the pyramidal cells.
Fig. 9 – The second of Cajal's intracortical and corticocortical “arcs”. This “intragriseal arc with intercalation of cells with
ascending axons” begins with terminations of thalamic fibers upon small pyramidal and stellate cells whose ascending axons
contact the dendritic tufts of layer III and V pyramidal cells. The collateral axons of these cells, as shown in Fig. 8, would carry
the activity to other pyramidal cells located in the same gyrus but at a considerable distance from the terminations of the
afferent fiber. This activity would be further extended by the intercalation of the axons of horizontal cells located in layer I
carrying activity to even more distant pyramidal cells, including those located in other gyri.
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Fig. 10 – The third of Cajal's intracortical and corticocortical “arcs”. This “intraareal intragriseal arc” is formed by pyramidal cells
whose axons terminate in the association areas of the same hemisphere, extending the influence of an afferent input to areas
concerned with higher level cortical function in the same hemisphere. An extension of this arc is the “interhemispheric arc”
whereby activity is carried across the corpus callosum by the projecting axons of the pyramidal cells.
system that had only been dimly glimpsed with earlier staining
techniques. In the course of his rather brief period of working on
the central nervous system, Golgi provided the details about
nerve cell structure, dendritic organization and axonal branching that served as the launching platform for Cajal's investigations and the fundamental basis on which Cajal was to build up
those patterns of intrinsic cerebral circuitry that have been
modified only in minor details to this day.
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